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Team:Queens Canada/Guide/Protein Structure
From 2012.igem.org
Guide to making Chimeric Proteins- Protein Expression
In our chimeras we have linker sequences on either side of the chimeric insert sequence. The linkers we chose to use were inert, and flexible linker sequences, with different sequences on either side so that overlap extension will be successful.
FliC -GGGGS- insert -GGSGG- FliC
It is important to also consider the flexible regions of the protein in which you are making your insertion. Flexible regions may be able to accommodate a protein insertion, and would not require very long linkers. However, longer linkers may be required to introduce enough flexibility for the inserted protein to fold properly.
The protein database is possibly the most important tool in chimeric protein design. Without having any previous knowledge of your structure, it is impossible to really say where or how you would design your chimeric protein. In our case, the crystal structure for E. coli flagellin is not available on the protein database. However, the structure for the flagellin of S. typhymurium was available and is very similar to the structure of E. coli flagellin. The PDB ID is 1UCU.
Additionally, some general assumptions can be made when searching for a good place to make an insertion. For example, the site that we chose to make our variable domain insertions is:
...AVTVANDGTVTMATG...
The insertion was made in between AVT and TVT, replacing the amino acid sequence VANDG. We needed to have some spacing in between the overlap regions for PCR overlap extension, which is why VANDG was replaced.
The presence of several threonine, alanine, valine and glycine residues is indicative of a loop region, which would make a good spot for an insertion. Additionally, when we used PCR to make the overlapping regions for the insertion, we amplified off of the already existing linker used for ovarlapping as a deletion. Hence our total linker was:
...AVTTTGGGGS-insert-GGSGGTSTVT...
First, you'll have to install it (don't worry, it's free): pymol.org
Pymol is a really useful program for viewing and modifying protein structures. Here we'll quickly go step by step into specifically building proteins in PYmol and what you need to know
- Load your protein in PYmol: under 'plugins' choose 'PDB loader service' and enter the 4-digit PDB ID for your desired protein.
- Right now you'll be in viewing mode, which lets you rotate and move the camera. To switch to "Editing mode" click on the box in the bottom right that reads "3-button viewing mode". This will switch you to editing mode.
- Here you'll have all the options for actually moving your protein around and rotating it.
- On the PYmol menu and script window, you can switch to "Builder". This will give you specific options for adding amino acids and other molecules to your structure.
- To make an insertion, you'll have to delete a bond. This is one of the options in the Builder window. Or you can delete a residue or residues, by selecting a residue in viewer mode, right clicking and choosing remove.
- Now you have free bonds where you can build residues. Simply click on the atom where you want to add a residue (usually a terminal N or C=O), then click on the residue you want to add in the Builder window. The default secondary structure is in a parallel beta sheet, which is essentially just a straight line.
- Rotating bonds can be done by clicking on an atom and then double-click and hold the part that you want to rotate. Remember, don't rotate between N and C=O bonds!
- The last and most important thing you'll need to know, which isn't obvious at first is how Pymol actually make bonds. It does it purely by proximity. So if you have atoms that are within 1.5 angstroms, Pymol will automatically make a bond between those two atoms after you save and reload the .pdb file.
- The code "distance (pk1),(pk2)" will measure the distance between two selected atoms. You may also use the measurement wizard to find distances and well as phi and psi angles.
- Move and twist your bonds and proteins to make your chimeric protein work!
